Sensores

In this study, the antibody and T cell mediated immune response following influenza vaccination was evaluated during two consecutive influenza seasons from 2009 to 2011. The emergence of A(H1N1)pdm09 provided us with the opportunity to evaluate influenza vaccine immunogenicity in a unique setting. The Dutch Health Council recommended vaccination with two doses of a MF59-adjuvanted monovalent A(H1N1)pdm09 vaccine, which allowed us to evaluate both the unusual two dose schedule and the effect of MF59 adjuvation on immunogenicity of the pandemic vaccine. One dose of the pandemic vaccine induced antibody responses sufficient for providing seroprotection and, in addition, induced vaccine- specific T cell responses. A second dose further increased antibody responses but not T cell responses.

Furthermore, in the subsequent influenza season, the trivalent seasonal vaccine contained the pandemic strain of the previous season, A(H1N1)pdm09, and a new H3N2 strain, A/Perth/16/2009(H3N2), allowing for analysis of booster effect of previous vaccination with the A(H1N1)pdm09 strain. Both antibody and T cell responses could be boosted by the seasonal vaccine. In addition, a comparison could be made of an adjuvanted and unadjuvanted influenza vaccine. Immunogenicity of the influenza vaccines was evaluated by measuring both vaccine-specific antibody and T cell responses during both influenza seasons. Furthermore, we show that the seasonal vaccine alone is capable of inducing vaccine-specific T cell responses, despite the fact that the vaccine did not contain an adjuvant.

In addition, residual antibody levels remained detectable for over 15 months, while T cell levels had reduced back to baseline levels by that time. We conclude that vaccine-induced antibody responses are detectable in the blood for a longer period than T cell responses measured in this study. However, this does not necessarily indicate that vaccine-specific T cells are no longer present. Memory T cells might reside in (lymphoid) tissues instead of in circulation, which is not reflected by measuring PBMC-specific T cell responses in the blood (23-25).

During the first season, immunogenicity of the MF59-adjuvanted monovalent A(H1N1)pdm09 vaccine was evaluated. Adjuvants, such as MF59, have been shown to reduce the dose of antigen needed and to induce a longer lasting antibody-mediated immune response (8). To assure seroprotection, the Dutch Health Council chose to advise a two-dose schedule as recommended by the manufacturer. The choice of administering two doses was based on studies on avian influenza vaccination where two doses were needed to obtain sufficient antibody responses (26). These studies with H5 influenza vaccines showed that two adjuvanted vaccine doses were required to obtain antibody levels that correlate with protection according to EMA criteria and furthermore they induced memory B cells (27- 30). In this study, we observed in a cohort of healthy individuals that one dose induced

antibody responses sufficient to conform to EMA guidelines for the registration of pandemic vaccines. In concordance, others have shown that one dose also induced adequate levels of seroprotection in other target groups of 2009 pandemic vaccination, i.e., infants, elderly and immunocompromised individuals (31-33). In addition, data on H9N2 vaccines indicate that one dose of an adjuvanted vaccine is sufficient for protection against H9N2 subtypes (34). Efficacy of vaccines for newly emerging subtypes appear to be affected by cross-reactive immunity. For individuals that do not have pre-existing immunity, one or even two doses might not be sufficient to provide seroprotective antibodies as shown by a study with H5 subtypes (27). In contrast, a study on H9N2 vaccines showed that individuals who had cross- reactive H2 antibodies available, responded better to one dose of an H9N2 subunit vaccine than individuals that did not have cross-reactive antibodies available. This cross-reactivity has been proposed to be due to structure similarity of H2 and H9 (35). However, there is also literature available on neutralizing antibodies that are directed to the conserved stalk domain of HA (36-38). Therefore, cross-reactive immunity may provide partial protection that can be boosted by vaccination. Thus, when an influenza subtype crosses over to the human population for the first time, the presence of cross-reactive immunity could determine whether one or two doses are needed to provide seroprotection.

Although antibodies provide primary protection against influenza virus infection, T cells are needed to clear infection when these antibodies fail to induce neutralizing protection. The importance of T cells is especially clear in situations where low cross-protective neutralizing antibodies are observed, and shows the additive value of inducing T cell responses by vaccination (39-41). The MF59-adjuvanted vaccine has been shown to induce follicular helper CD4+ _{T cells and presence of these cells predict antibody responses (42). Furthermore, MF59} recruits immune cells, such as macrophages and monocytes, to the site of infection, and was shown to induce differentiation of monocytes to DCs, which in turn can also prime CD8+ _T cell responses (43). Therefore, the MF59-adjuvanted vaccine is expected to induce T cell responses in addition to antibody responses.

Analysis of vaccine-induced T cell responses was performed by stimulation of PBMCs with whole influenza virus or HA or NA-specific peptide pools using an IFN-γ ELISpot. In most individuals, we observe a background level of T cell responses before vaccination, which are more prominent in the whole virus stimulation assays. Background levels of these responses are the consequence of activation of T cells induced by natural infection or previous vaccination and will include the response to internal viral proteins. In the model, we correct for these background levels by studying an additional induction. Peptide pools solely containing vaccine antigens enabled us to make assumptions about vaccine-induced T cells alone. However, future studies are required to analyze the full cytokine profile of these responses, dissecting the nature of adjuvanted and unadjuvanted vaccine-induced T cells.

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In the peptide ELISpot assay, we observed an induction of T cells already after vaccination with one dose of the adjuvanted vaccine. However, after the second dose, T cell responses remained similar to responses measured after one dose. McElhany et al. even found a negative correlation between antibody levels and cytokine ratios in elderly and proposed that a second dose might skew T cell responses to the production of IL-10, which limits CTL induction but is advantageous for antibody responses (44). We only evaluated T cell responses by IFN-γ production and are therefore currently not able to support this notion. Others reported an inverse correlation between pre-vaccination IFN-γ production and the magnitude of responses post-vaccination (45, 46). As described by Bodewes et al., annual vaccination with a seasonal vaccine hampers the development of influenza-specific CD8+ _T cells in children, indicating that vaccination history also affects the development of T cell responses (47). To conclude, both a second dose as well as previous vaccination and exposure to influenza might affect T cell responses induced by vaccination.

The number of doses and the quantity of antigen that are needed to induce sufficient protection during a pandemic might be related to the presence of cross-reactive antibody and T cell immunity. It is therefore important to obtain knowledge on pre-existing immunity to the virus, since this can be an indication whether a second dose is necessary. During the 2009 H1N1 pandemic, data became available that individuals had some cross-reactive T cells available that provided partial protection (48). In addition, antibodies cross-reacting to the pandemic strain were observed in older adults, which corresponds with the lower number of affected individuals in this age group (49). Although in this study individuals born during the previous H1N1 era, from 1917-1956, were excluded to limit the effects of cross-reactive immunity on the measurement of vaccine efficacy, there may still have been cross-reactive immunity present in younger individuals, which may in part explain why one dose of the pandemic vaccine already induced sufficient protection.

Therefore, it is also of significance what type of immune response is induced by regular seasonal vaccines, especially if administration of a second dose or annual vaccination might have a negative effect on the T cell response that is induced. In this study, we showed that both the adjuvanted pandemic vaccine containing 7.5 µg HA and the unadjuvanted seasonal vaccine containing 15 µg of HA were capable of inducing T cell responses. Others have shown that T cell responses can be induced by unadjuvanted split seasonal influenza vaccination in children, but focused only on internal influenza proteins (50). To date, not much data is available on the induction of T cells by the trivalent inactivated influenza vaccine containing HA and NA as viral antigens. However, we show that even an unadjuvanted subunit vaccine is capable of inducing T cell responses.

This study has some limitations. During this study, individuals were monitored for influenza- like illness (ILI) and ILI cases were laboratory confirmed for the presence of influenza within 72 hours of onset of symptoms. However, both the pandemic and consecutive year were

very mild influenza seasons in the Netherlands and only sporadic infections were observed in individuals in this study. Therefore, we could correct in our model for influenza infections during the study period. In addition, individuals with a laboratory-confirmed A(H1N1)pdm09 influenza infection before the start of the study were excluded. However, we cannot exclude the possibility that subclinical infections have occurred. Another confounding factor is the limited number of individuals that were enrolled in the CV group, which may have affected results of the HA and NA ELISpot assay, specifically. In addition, in this study, IFN-γ was used as the only read-out for T cell responses, while other cytokines or assays may provide with a more complete picture of the T cell response. For example, it would be interesting to elucidate whether the T cell responses measured in this study can be contributed to CD4+ _T cells, CD8+ _{T cells or both and the additional cytokines secreted by the activated T cells.} Summarizing, we showed that one dose of the MF59-adjuvanted pandemic vaccine induced seroprotective levels of antibodies, which were boosted after administration of a second dose. This second dose did not boost the number of vaccine-induced T cell responses. At the start of the second season, a residual level of antibody and T cell levels was detectable in individuals vaccinated in the previous season. Administration of the 2010-2011 seasonal vaccine boosted both antibody and T cell levels. Comparison of the adjuvanted and unadjuvanted vaccine showed that the adjuvanted vaccine induced significantly higher antibody levels, while T cell levels induced after pandemic or seasonal vaccination were similar. Furthermore, we show that antibody levels were still detectable after 15 months, whereas T cell levels had decreased back to baseline.

These findings have key implications for influenza vaccination strategies, especially during pandemic situations. When cross-protective immunity is available, in the form of conserved antibody or T cell responses, one vaccination dose might be sufficient to provide protection. Since repeated influenza vaccination may not be favorable for the induction of T cell responses, it is important to have knowledge on cross-reactive immunity available. Therefore, studies describing the immune response following influenza vaccination should not only focus on the humoral immune response, but should also include analysis of cellular responses.

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